Methods and compositions for reversing or mitigating skin aging

ABSTRACT

A method for reversing or mitigating skin aging comprising topically applying a composition which comprises at least one oxidant, and at least one fluorescent dye capable of activating the oxidant and illuminating said composition for a time sufficient to activate the at least one oxidant.

BACKGROUND (a) Field

The subject matter disclosed generally relates to a skin rejuvenationcomposition and method of treatments thereof.

(b) Related Prior Art

The application of non-ablative skin rejuvenation to repair, or offset,the results of both chronological- and photo-ageing in the skin of theface, neck, hands and exposed areas of the body has become extremelypopular. From the original mechanical and chemical peels, cliniciansrapidly adopted the use of ablative skin rejuvenation using lasers,albeit with the negative effects of severe morbidity (erythema andedema) resulting in patient downtime. These disadvantages significantlyoffset the good results of the treatment. Photorejuvenation techniquessuch as lasers and intense pulsed light (IPL) sources were thendeveloped to deliver thermal damage to the dermis under cooling, termednon-ablative, skin rejuvenation. This generally localised thermal damageto the deeper layers of the skin (dermis) whilst the forced coolinghelped to protect the upper (and therefore highly visible) layers fromthermal damage. Laser ablative skin resurfacing has been a popularmodality for the removal or improvement of major wrinkles and othersevere symptoms of aging. The principles of ablative therapy are basedon light-tissue interaction delivering the optimum amount of controlledresidual thermal damage with precise epidermal ablation, thereforeinvoking a wound response and thus maximizing the clinical result whilstminimizing side effects and their associated downtime. Unfortunately,the resulting crusting, oedema and long-term erythema are majorstumbling blocks for all but the most determined patient.

Photodynamic therapy of the skin using photoactive dyes such asErythrosine B, Safranin O has been employed to destroy bacteria, asdescribed in WO 05/032459 and WO 05/021094, both to Albrecht et al. Thephotoactive dyes are employed to directly destroy bacteria. Thecompositions described in these patent applications lack oxidants andhealing factors, and they are not employed for directly promoting skinrejuvenation.

Hence, there is a need to accelerate and/or improve rejuvenation of theskin following invasive or non-invasive treatment. Accelerated recoveryoffers enhanced patient safety (e.g. a reduced window for infection andpain) as well as fitting with the trend towards less aggressive and lessinvasive treatments.

Therefore, it would be highly desirable to be provided with a novelcomposition for the rejuvenation of skin damages in order to improve apatient's skin condition and accelerate the healing process followingthe establishment of lesions caused by chronological ageing andphoto-ageing.

SUMMARY

In accordance with one embodiment there is disclosed a skin rejuvenationcomposition which comprises at least one oxidant, at least onephotoactivator capable of activating the oxidant, and at least onehealing factor chosen from hyaluronic acid, glucosamine and allantoin,in association with a pharmaceutically acceptable carrier.

The oxidants may be chosen from hydrogen peroxide, carbamide peroxideand benzoyl peroxide.

The skin rejuvenation composition may further comprise at least onehydrophilic gelling agent.

The hydrophilic gelling agent may be chosen from glucose, modifiedstarch, methyl cellulose, carboxymethyl cellulose, propyl cellulose,hydroxypropyl cellulose, Carbopol® polymers, alginic acid, sodiumalginate, potassium alginate, ammonium alginate, calcium alginate, agar,carrageenan, locust bean gum, pectin, gelatin.

The photoactivators may be chosen from a xanthene derivative dye, an azodye, a biological stain, and a carotenoid.

The xanthene derivative dye may be chosen from a fluorene dye, afluorone dye, and a rhodole dye.

The fluorene dye may be chosen from a pyronine dye and a rhodamine dye.

The pyronine dye may be chosen from pyronine Y and pyronine B.

The rhodamine dye may be chosen from rhodamine B, rhodamine G andrhodamine WT.

The fluorone dye may be chosen from fluorescein and fluoresceinderivatives.

The fluorescein derivative may be chosen from phloxine B, rose bengal,and merbromine.

The fluorescein derivative may be chosen from eosin and erythrosine.

The azo dye may be chosen from methyl violet, neutral red, para red,amaranth, carmoisine, allura red AC, tartrazine, orange G, ponceau 4R,methyl red, and murexide-ammonium purpurate.

The biological stain may be chosen from saffranin O, basic fuchsin, acidfuschin, 3,3′ dihexylocarbocyanine iodide, carminic acid, andindocyanine green.

The carotenoid may be chosen from crocetin, α-crocin(8,8-diapo-8,8-carotenoic acid), zeaxanthine, lycopene, α-carotene,β-carotene, bixin, and fucoxanthine.

The carotenoid may be present in the composition as a mixture chosenfrom saffron red powder, annatto extract and brown algae extract.

The skin rejuvenation composition may further comprise at least onechelating agent.

The chelating agent may be chosen from ethylenediaminetetraacetic acid(EDTA) and ethylene glycol tetraacetic acid (EGTA).

The skin rejuvenation composition may further comprise at least onelipolysis stimulating factor.

The lipolysis stimulating factor may be chosen from caffeine andparaxanthine.

In accordance with one embodiment, there is disclosed a method for skinrejuvenation which comprises the steps of a) topically applying on apatient's skin a composition comprising at least one oxidant, at leastone photoactivator capable of activating the oxidant; and b) treatingsaid skin of step a) to actinic light for a time sufficient for saidphotoactivator to cause activation of said oxidant.

The method for skin rejuvenation may comprise exposing the skin toactinic light for a period of about 60 seconds to about 5 minutes.

The method for skin rejuvenation may comprise exposing the skin toactinic light for a period of about 60 seconds to about 5 minutes percm² of an area to be treated.

The method for skin rejuvenation may comprise exposing the skin to asource of actinic light that is in continuous motion over the area beingtreated.

The method for skin rejuvenation may comprise exposing the skin toactinic light that may be visible light having a wavelength between 400nm and 600 nm.

The following terms are defined below.

The term “hydrophilic gelling agent” is intended to mean a material thatthickens and stabilizes liquid solutions, emulsions, and suspensions.Hydrophillic gelling agents dissolve in liquid and provide a structuregiving the resulting gel an appearance of a solid matter, while beingmostly composed of a liquid. Hydrophillic gelling agents are verysimilar to thickeners.

The term “actinic light” is intended to mean light energy emitted from aspecific light source (lamp, LED, or laser) and capable of beingabsorbed by matter (e.g. the photoactivator defined below) and producean identifiable or measurable change when it interacts with it; asclinically identifiable change we can presume a change in the color ofthe photoactivator used (e.g. from red to transparent).

The term “photoactivator” is intended to mean a chemical compoundcapable of absorbing actinic light. The photoactivator readily undergoesphotoexcitation and then transfers its energy to other molecules, thusenhancing or accelerating the dispersion of light, and enhancing oractivating the oxidant present in the reaction mixture.

The term “oxidant” is intended to mean a either a chemical compound thatreadily transfers oxygen atoms and oxidize other compounds, or asubstance that gains electrons in a redox chemical reaction.

The term “chelating agent” is intended to mean a chemical that removesmetal ions, such as iron, and holds them in solution.

The term “healing factor” is intended to mean a compound that promotesor enhances the healing or regenerative process of a tissue.

The term “lipolysis” is intended to mean the process in which lipids arebroken down into their constituent fatty acids.

The term “time of exposure to actinic light” is intended to mean thetime a tissue, skin or wound is exposed to actinic light per applicationof actinic light.

The term “total time of exposure to actinic light” is intended to meanthe cumulative time a tissue, skin or wound is exposed to actinic lightafter several application of actinic light.

The term “pharmaceutically acceptable carrier” is intended to mean apreservative solution, a saline solution, an isotonic (about 0.9%)saline solution, or about a 5% albumin solution, suspension, sterilewater, phosphate buffered saline, and the like. Other buffering agents,dispersing agents, and inert non-toxic substances suitable for deliveryto a patient may be included in the compositions of the presentinvention. The compositions may be solutions, suspensions or anyappropriate formulation suitable for administration, and are typicallysterile and free of undesirable particulate matter. The compositions maybe sterilized by conventional sterilization techniques.

The term “rejuvenation” is intended to mean is the reversal ormitigation of the aging process, or any other processes (e.g. abrasioncaused by a fall, burns, etc.) that may have damaged or caused anaccumulation of damage to macromolecules, cells, tissues and organs,including the skin. Rejuvenation is the repair of any of such damage.

The term “photorejuvenation” is intended to mean the use of light forthe reversal or mitigation of the aging process, or any other processes(e.g. abrasion caused by a fall, burns, etc.) that may have damaged orcaused an accumulation of damage to macromolecules, cells, tissues andorgans, including the skin.

Features and advantages of the subject matter hereof will become moreapparent in light of the following detailed description of selectedembodiments, as illustrated in the accompanying figures. As will berealized, the subject matter disclosed and claimed is capable ofmodifications in various respects, all without departing from the scopeof the claims. Accordingly, the drawings and the description are to beregarded as illustrative in nature, and not as restrictive and the fullscope of the subject matter is set forth in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that Eosin Y does not affect cellular viability. HepG2 cells were treated for 24 hours with increasing concentrations (0.001to 100 μM) of Eosin Y, or left untreated (CTL). Staurosporine (STS) wasused as a positive control inducing cellular mortality. Higherconcentrations (0.5 and 1 mM) of Eosin Y could not be tested as the dyeinterfered with the assay.

FIG. 2 illustrates that Erythrosine B does not affect cellularviability. Hep G2 cells were treated for 24 hours with increasingconcentrations (0.001 to 100 μM) of Erythrosine B, or left untreated(CTL). Staurosporine (STS) was used as a positive control inducingcellular mortality. Higher concentrations (0.5 and 1 mM) of ErythrosineB could not be tested as the dye interfered with the assay.

FIG. 3. Illustrates a transversal view of rat skin treated or not withthe composition according to the present invention in conjunction withexposure to light.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with one embodiment, there is provided a skin rejuvenationcomposition and the method of use of the composition on a patient'sskin. This product accelerate rejuvenation of a skin.

In accordance with another embodiment, there is provided a method of useof the composition that is a photodynamic technique whereby thecomposition is activated by light, providing a beneficial effect on theskin and promoting rejuvenation.

The composition and method may be used to treat damages caused by normaltemporal aging, as well as premature aging of the skin caused byexposure to the sun. These damages include but are not limited tophoto-aging, hyperpigmentation, large pores, skin irregularities, finelines, dark skin discoloration, sun damage, pigmented lesions (e.g.melanocytic proliferations, reticular melanotic hypermelanoses),hyperpigmented lesions (e.g. post-traumatic hyperpigmentation),wrinkles, sagging skin, and scars.

It may also be used to treat a wide array of skin conditions. Theseinclude but are not limited to acne and acne scars, rosacea. Nevi(blemish on the skin), Mongolian spots, such as Becker's nevus, bluenevus, congenital nevus, pigmented nevus (mole), nevus of Ota and Ito,pigmented spindle cell nevus, and dysplastic nevus may also be treatedwith the composition.

Visible vascular lesions to the skin may also be treated with thecomposition. These include but are not limited to matted telangiectasia,lentigines, cherry angioma (a.k.a. “De Morgan spots,” and “Senileangiomas), spider angioma (a.k.a. nevus araneus, spider nevus, orvascular spider), vascular lesions such as vascular birthmarks, blue,red or purple port wine stains, red or blue facial and leg veins (a.k.a.spider veins). The composition and method may be used to help in theremoval of most tattoos.

The composition comprises a number of active principles selected fromgroups of possible components. These various active principles each havetheir mechanism of action.

Oxidants

The composition comprises oxidants as a source of oxygen radicals.Peroxide compounds are oxidants that contain the peroxy group (R—O—O—R),which is a chainlike structure containing two oxygen atoms, each ofwhich is bonded to the other and a radical or some element. Suitableoxidants for preparation of the active medium include, but are notlimited to:

Hydrogen peroxide (H₂O₂) is the starting material to prepare organicperoxides. H₂O₂ is a powerful oxidizing agent, and the unique propertyof hydrogen peroxide is that it breaks down into water and oxygen anddoes not form any persistent, toxic residual compound. Hydrogen peroxidefor use in this composition can be used in a gel, for example with 6%hydrogen peroxide. A suitable range of concentration over which hydrogenperoxide can be used in the present composition is from about 3.5% toabout 6%.

Urea hydrogen peroxide (also known as urea peroxide, carbamide peroxideor percarbamide) is soluble in water and contains approximately 35%hydrogen peroxide. Carbamide peroxide for use in this composition can beused as a gel, for example with 16% carbamide peroxide that represents5.6% hydrogen peroxide. A suitable range of concentration over whichurea peroxide can be used in the present composition is from about 10%to about 16%. Urea peroxide brakes down to urea and hydrogen peroxide ina slow-release fashion that can be accelerated with heat orphotochemical reactions. The released urea [carbamide, (NH₂)CO₂)], ishighly soluble in water and is a powerful protein denaturant. Itincreases solubility of some proteins and enhances rehydration of theskin and/or mucosa.

Benzoyl peroxide consists of two benzoyl groups (benzoic acid with the Hof the carboxylic acid removed) joined by a peroxide group. It is foundin treatments for acne, in concentrations varying from 2.5% to 10%. Thereleased peroxide groups are effective at killing bacteria. Benzoylperoxide also promotes skin turnover and clearing of pores, whichfurther contributes to decreasing bacterial counts and reduce acne.Benzoyl peroxide breaks down to benzoic acid and oxygen upon contactwith skin, neither of which are toxic. A suitable range of concentrationover which benzoyl peroxide can be used in the present composition isfrom about 2.5% to about 5%.

Inclusion of other forms of peroxides (e.g. organic or inorganicperoxides) should be avoided due to their increased toxicity and theirunpredictable reaction with the photodynamic energy transfer.

Photoactivators:

The photoactivators transfer light energy to the oxidants. Suitablephotoactivators can be fluorescent dyes (or stains), although other dyegroups or dyes (biological and histological dyes, food colorings,carotenoids) can also be used. Combining photoactivators may increasephoto-absorption by the combined dye molecules and enhance absorptionand photo-biomodulation selectivity. This creates multiple possibilitiesof generating new photosensitive, and/or selective photoactivatormixtures.

An advantageous characteristic of a photoactivator is increasedfluorescence. In the present invention, re-emission of light in thegreen to yellow spectrum would be advantageous, since it is a deeppenetrating wavelength range, with deep absorption by the blood. Thisconfers a strong increase on the blood flow, vasodilatation andangiokinetic phenomena. Suitable photoactivators include, but are notlimited to:

Xanthene Derivatives:

The xanthene derivative dyes have been used and tested for a long timeworldwide. They display low toxicity and increased fluorescence. Thexanthene group consists of 3 sub-groups that are: a) the fluorenes; b)fluorones; and c) the rhodoles.

The fluorenes group comprises the pyronines (e.g. pyronine Y and B) andthe rhodamines (e.g. rhodamine B, G and WT). Depending on theconcentration used, both pyronines and rhodamines may be toxic and theirinteraction with light may lead to increased toxicity. Similar effectsare known to occur for the rhodole dye group.

The fluorone group comprises the fluorescein dye and the fluoresceinderivatives.

Fluorescein is a fluorophore commonly used in microscopy with anabsorption max. of 494 nm and an emission max. of 521 nm. The disodiumsalt of fluorescein is known as D&C Yellow 8. It has very highfluorescence but photodegrades quickly. In the present composition,mixtures of fluorescein with other photoactivators such as indocyaningreen and/or saffron red powder will confer increased photoabsorption tothese other compounds.

Eosins group comprises Eosin Y (tetrabromofluorescein, acid red 87, D&CRed 22) with an abs. max 514-518 nm, stains cytoplasm of cells,collagen, muscle fibers and red blood cells intensely red; and Eosin B(acid red 91, eosin scarlet, dibromo-dinitrofluorescein), with the samestaining characteristics as Eosin Y. Eosin Y, eosin B, or a mixture ofboth can be used because of their sensitivity to the light spectra used:broad spectrum blue light, blue to green light and green light. Theirtissue and biofilm staining properties and their low toxicity are alsoadvantageous. Both eosin Y and eosin B stain red blood cells and thusconfer to the composition of the present invention haemostatic (controlsthe flow or stops the flow of blood) properties as well as increase theselective targeting of light to the soft tissues of the lesion or woundduring the application of the composition.

Phloxine B (2,4,5,7 tetrabromo 4,5,6,7,tetrachlorofluorescein, D&C Red28, acid red 92) is a red dye derivative of fluorescein which is usedfor disinfection and detoxification of waste water throughphotooxidation. It has an abs. max. of 535-548 nm. It is also used as anintermediate for making photosensitive dyes and drugs.

Erythrosine B (acid red 51, tetraiodofluorescein) is a cherry-pink,coal-based fluorine food dye used as a biological stain, and a biofilmand dental plaque disclosing agent, with max. abs. 524-530 nm in aqueoussolution. It is subject to photodegradation. Erythrosine is also used insome embodiments due to its photosensitivity to the light spectra usedand its ability to stain biofilms. Inclusion of erythrosine should befavored when using the composition in deep pockets of infected orcontaminated tissue, such as periodontal pockets in periodontal therapy.

Rose Bengal (4,5,6,7 tetrachloro 2,4,5,7 tetraiodofluorescein, acid red94) is a bright bluish-pink biological dye with an absorption max. of544-549 nm, that has been used as a dye, biological stain and diagnosticaid. Also used in synthetic chemistry to generate singlet from tripletoxygen.

Merbromine (mercurochrome) is an organo-mercuric disodium salt offluorescein with an abs. max. of 508 nm. It is used as an antiseptic.

Azo Dyes:

The azo (or diazo-) dyes share the N—N group, called azo the group. Theyare used mainly in analytical chemistry or as food colorings and are notfluorescent. Suitable azo dyes include: Methyl violet, neutral red, parared (pigment red 1), amaranth (Azorubine S), Carmoisine (azorubine, foodred 3, acid red 14), allura red AC (FD&C 40), tartrazine (FD&C Yellow5), orange G (acid orange 10), Ponceau 4R (food red 7), methyl red (acidred 2), murexide-ammonium purpurate.

Biological Stains:

Dye molecules commonly used in staining protocols for biologicalmaterials can also be used as photoactivators. Suitable biologicalstains include:

Saffranin (Saffranin O, basic red 2) is also an azo-dye and is used inhistology and cytology. It Is a classic counter stain in a Gram stainprotocol.

Fuchsin (basic or acid) (rosaniline hydrochloride) is a magentabiological dye that can stain bacteria and has been used as anantiseptic. It has an abs. max. 540-555 nm.

3,3′ dihexylocarbocyanine iodide (DiOC6) is a fluorescent dye used forstaining cell's endoplasmic reticulum, vesicle membranes andmitochondria. It shows photodynamic toxicity; when exposed to bluelight, has a green fluorescence.

Carminic acid (acid red 4, natural red 4) is a red glucosidalhydroxyanthrapurin naturally obtained from cochineal insects.

Indocyanin green (ICG) is used as a diagnostic aid for blood volumedetermination, cardiac output, or hepatic function. ICG binds stronglyto red blood cells and when used in mixture with fluorescein, itincreases the absorption of blue to green light.

Carotenoids

Carotenoid dyes can also act as photoactivators.

Saffron red powder is a natural carotenoid-containing compound. Saffronis a spice derived from Crocus sativus. It is characterized by a bittertaste and iodoform or hay-like fragrance; these are caused by thecompounds picrocrocin and saffranal. It also contains the carotenoid dyecrocin that gives its characteristic yellow-red color.

Saffron contains more than 150 different compounds many of them arecarotenoids: mangicrocin, reaxanthine, lycopene, and various α- andβ-carotenes, that show good absorption of light and beneficialbiological activity. Also saffron can act as both a photon-transferagent and a healing factor. Saffron color is primarily the result ofα-crocin (8,8 diapo-8,8-carotenoid acid). Dry saffron red powder ishighly sensitive to fluctuating pH levels and rapidly breaks downchemically in the presence of light and oxidizing agents. It is moreresistant to heat. Data show that saffron has anticarcinogenic,immunomodulating and antioxidant properties. For absorbance, it isdetermined for the crocin specific photon wavelength of 440 nm (bluelight). It has a deep red colour and forms crystals with a melting pointof 186° C. When dissolved in water it forms an orange solution.

Crocetin is another compound of saffron that was found to express anantilipidemic action and promote oxygen penetration in differenttissues. More specifically it was observed an increased oxygenation ofthe endothelial cells of the capillaries. An increase of the oxygenationof muscles and cerebral cortex was observed and led to an increasedsurvival rate in laboratory animals with induced hemorrhagic shock oremphysema.

Anatto a spice contains as main constituent (70-80%) the carotenoidbixin which displayed relevant antioxidative properties.

β-carotene, also displayed suitable characteristics.

Fucoxanthine is a constituent of brown algae with a pronounced abilityfor photosensitization of red-ox reactions.

Healing Factors:

Healing factors comprise compounds that promote or enhance the healingor regenerative process of the tissues on the application site of thecomposition. During the photoactivation of the composition, there is anincrease of the absorption of molecules at the treatment site by theskin or the mucosa. An augmentation in the blood flow at the site oftreatment is observed for an extent period of time. An increase in thelymphatic drainage and a possible change in the osmotic equilibrium dueto the dynamic interaction of the free radical cascades can be enhancedor even fortified with the inclusion of healing factors. Suitablehealing factors include, but are not limited to:

Hyaluronic acid (Hyaluronan, hyaluronate): is a non-sulfatedglycosaminoglycan, distributed widely throughout connective, epithelialand neural tissues. It is one of the primary components of theextracellular matrix, and contributes significantly to cellproliferation and migration. Hyaluronan is a major component of theskin, where it is involved in tissue repair. While it is abundant inextracellular matrices, it contributes to tissues hydrodynamics,movement and proliferation of cells and participates in a wide number ofcell surface receptor interactions, notably those including primaryreceptor CD44. The hyaluronidases enzymes degrade hyaluronan. There areat least seven types of hyaluronidase-like enzymes in humans, several ofwhich are tumor suppressors. The degradation products of hyaluronicacid, the oligosaccharides and the very-low molecular weight hyaluronicacid, exhibit pro-angiogenic properties. In addition, recent studiesshow that hyaluronan fragments, but not the native high molecular massof hyaluronan, can induce inflammatory responses in macrophages anddendritic cells in tissue injury. Hyaluronic acid is well suited tobiological applications targeting the skin. Due to its highbiocompatibility, it is used to stimulate tissue regeneration. Currentstudies evidenced hyaluronic acid appearing in the early stages ofhealing to physically create room for white blood cells that mediate theimmune response. It is used in the synthesis of biological scaffolds forwound healing applications and in wrinkle treatment.

Glucosamine: is one of the most abundant monosaccharides in humantissues and a precursor in the biological synthesis of glycosilatedproteins and lipids. It is commonly used in the treatment ofosteoarthritis. The common form of glucosamine used is its sulfate salt.Glucosamine shows a number of effects including an anti-inflammatoryactivity, stimulation of the synthesis of proteoglycans and thesynthesis of proteolytic enzymes. A suitable range of concentration overwhich glucosamine can be used in the present composition is from about1% to about 3%.

Allantoin: is a diureide of glyosilic acid. It has keratolytic effect,increases the water content of the extracellular matrix, enhances thedesquamation of the upper layers of dead (apoptotic) skin cells, andpromotes skin proliferation and wound healing.

Also, saffron can act as both a photon-transfer agent and a healingfactor.

Chelating Agents:

Chelating agents can be included to promote smear layer removal inclosed infected pockets and difficult to reach lesions; act as a metalion quencher and as a buffer. Suitable chelating agents include, but arenot limited to:

Ethylenediaminotetraacetic acid (EDTA): It is an aminoacid, used tosequester di- and trivalent metal ions. EDTA binds to metals via 4carboxylate and 2 amine groups. EDTA forms especially strong complexeswith Mn(III), Fe(III), Cu(III), Co(III). Prevents collection of theplatelets and blood clots formation. It is used in the endodontictherapy as a smear layer removal agent during instrumentation. It isused to buffer solutions.

Ethylene glycol tetraacetic acid (EGTA) is related to EDTA, but with amuch higher affinity for calcium than for magnesium ions. It is usefulfor making buffer solutions that resemble the environment inside livingcells and is often employed in dentistry, more specifically endodontics,in the removal of smear layer.

Lipolysis Stimulating Factors:

Lipolysis stimulating factors can be included for use of the compositionin cosmetic applications, such as wrinkle treatment.

Caffeine, and the metabolic derivative of caffeine paraxanthine canincrease in the lipolysis process to releases glycerol and fatty acidsinto the blood stream.

Hydrophilic Gelling Agents

The skin rejuvenation composition may also contain one or morehydrophilic gelling agent. The hydrophilic gelling agent enhances theconsistency of the composition and contributes to facilitating theapplication of the composition to the skin or wounded area. Also, whenused with hydrogen peroxide (H₂O₂), it may contribute to the slow therelease of the H₂O₂, and provide a more immediate reaction because pureH₂O₂ can be used directly. Suitable hydrophilic gelling agent include,but are not limited to glucose, modified starch, methyl cellulose,carboxymethyl cellulose, propyl cellulose, hydroxypropyl cellulose,Carbopol® polymers, alginic acid, sodium alginate, potassium alginate,ammonium alginate, calcium alginate, agar, carrageenan, locust bean gum,pectin, and gelatin.

Use of the Composition

The inclusion of suitable photosensitive compounds and activation with alight source of a proper wavelength, leads to the acceleration in thebreakdown process of the source of peroxide (the oxidant) and the otherreactions that take place, via a photodynamic phenomenon. The includeddyes are illuminated by photons of a certain wavelength and excited to ahigher energy state. When the photoactivators' excited electrons returnto a lower energy state, they emit photons with a lower energy level,thus causing the emission of light of a longer wavelength (Stokesshift). In the proper environment, much of this energy transfer istransferred to oxygen or the reactive hydrogen peroxide and causes theformation of oxygen radicals, such as singlet oxygen.

The singlet oxygen and other reactive oxygen species generated by theactivation of the composition are thought to operate in a hormeticfashion. That is, a health beneficial effect is brought about by the lowexposure to a normally toxic stimuli (e.g. reactive oxygen), bystimulating and modulating stress response pathways in cells of thetargeted tissues. Endogenous response to exogenous generated freeradicals (reactive oxygen species) is modulated in increased defensecapacity against the exogenous free radicals and induces acceleration ofhealing and regenerative processes. Furthermore, activation of thecomposition will also produce an antibacterial effect. The extremesensitivity of bacteria to exposure to free radicals makes thecomposition of the present invention a de facto bactericidalcomposition.

Possible mechanism of action should be a fortified redox signalingphenomenon resulting in accentuated signal transduction process in whichcells convert one kind of signal into another; activated “secondmessengers” induce a “signal cascade” beginning with a relatively smallstimulus that elicits a large response via biologically monitoredamplification of such signals. These complex mechanisms act possiblyinvolving angiogenic phenomena via growth factor activation.

This method could be described as a form of photodynamic therapy.However, unlike other photodynamic techniques, where thephotoactoactivators are incorporated in the tissue structure, in thepresent method, the photoactive material is in simple contact with thetissue and acts when activated by light, as a “photodynamic device” thatchemically interacts with the tissue. Additionally, the actinic lightpenetrates the tissue, and the light that is emitted by thephotoactivator (light of a longer wavelength) is also absorbed by thetissue.

Any source of actinic light can be used. Any type of halogen, LED orplasma arc lamp, or laser may be suitable. The primary characteristic ofsuitable sources of actinic light will be that they emit light in awavelength (or wavelengths) appropriate for activating the one or morephotoactivators present in the composition. In one embodiment, an argonlaser is used. In another embodiment, a potassium-titanyl phosphate(KTP) laser (e.g. a Greenlight™ laser) is used. In yet anotherembodiment, a LED photocuring device is the source of the actinic light.In yet another embodiment, the source of the actinic light is a sourceof visible light having a wavelength between 400 and 600 nm.Furthermore, the source of actinic light should have a suitable powerdensity. Suitable power density for non-collimated light sources (LED,halogen or plasma lamps) are in the range from about 900 mW/cm² to about2000 mW/cm². Suitable power density for laser light sources are in therange from about 0.5 mW/cm² to about 0.8 mW/cm².

The duration of the exposure to actinic light will be dependent on thesurface of the treated area, and on the type of lesion, trauma or injurythat is being treated. The photoactivation of the composition may takeplace within seconds or even fragment of seconds, but a prolongedexposure period is beneficial to exploit the synergistic effects of theabsorbed, reflected and reemitted light on the composition of thepresent invention and its interaction with the tissue being treated. Inone embodiment, the time of exposure to actinic light of the tissue,skin or wound on which the skin rejuvenation composition has beenapplied is a period between 60 second and 5 minutes. In anotherembodiment, the time of exposure to actinic light of the tissue, skin orwound on which the skin rejuvenation composition has been applied is aperiod between 60 seconds and 5 minutes per cm² of the area to betreated, so that the total time of exposure of a 10 cm² are would bebetween 10 minutes and 50 minutes. In yet another embodiment, the sourceof actinic light is in continuous motion over the treated area for theappropriate time of exposure. In yet another embodiment, multipleapplications of the skin rejuvenation composition and actinic light areperformed. In some embodiments, the tissue, skin or wound is exposed toactinic light at least two, three, four, five or six times. In someembodiments, a fresh application of the skin rejuvenation composition isapplied before exposure to actinic light.

ALTERNATIVE EMBODIMENTS Example I

An exemplary skin rejuvenation composition was prepared by mixing thefollowing components:

Oxidant Photoactivators Healing factor(s) Carbamide Erythrosine B (0.5%)Glucosamine sulfate (3%) peroxide (16%) Eosin B (0.25%) Hyaluronic acid(3%) Saffron Red powder (0.25%)

The oxidant (4 mL) and healing factors (1.5 mL) were mixed and thecombined with the photoactivators (1 mL). The resulting composition wasapplied to the skin of a patient, and activated with actinic lightprovided by a LED photocuring device (blue light). The composition wasremoved following treatment.

Example II

An second exemplary skin rejuvenation composition was prepared by mixingthe following components:

Oxidant Photoactivators Healing factor(s) Carbamide FluoresceinGlucosamine sulfate (3%) peroxide (16%) Indocyanin green Hyaluronic acid(3%) Saffron Red powder (0.25%)

The oxidant (4 mL) and healing factors (1.5 mL) were mixed and thecombined with the photoactivators (1 mL). The resulting composition wasapplied to the skin of a patient, and activated with actinic lightprovided by a LED photocuring device (blue light). The composition wasremoved following treatment.

This second exemplary composition is using the fluorescein dye as aphotoactivator to other dyes (indocyanine green and saffron red powder)present in the composition. The addition of a small amount offluorescein to the indocyanine green and saffron red powder solutioncaused reemission of light at wavelengths that activated the other dyecompounds and improved the treatment by increasing the establishedclinical absorption/reemission criteria.

Indocyanine green binds well to hemoglobin and helps the selectiveenergy absorption by the tissues and also helps targeting these tissueswith the generated free radical cascades. Also, this photoactivatorsmixture is able to render saffron red fluorescent, which again improvesboth the photodynamic and biostimulating phenomena.

Example III

The toxicity of the photoactivators Eosin Y and Erythrosine B wasevaluated by measuring the cytotoxicity of these compounds on humancells. Hep G2 human hepatocellular carcinoma cells with an epithelialmorphology were treated for 24 hours with increasing concentrations(0.001 to 100 μM) of Eosin Y or Erythrosine B, and the cellular survivalwas evaluated. Increasing concentrations of either Eosin Y (FIG. 1) orErythrosine B (FIG. 2) did not affect cellular viability when comparedto untreated cells. Staurosporine (STS) was used as a positive controlfor inducing cellular mortality and caused a dose-dependent effect(FIGS. 1 and 2). Similar results were obtained by measuring cell deathby release of lactate dehydrogenase (LDH). Therefore, neither Eosin Y orErythrosine B caused increased cellular mortality.

Example IV

Skin Rejuvenation in an Animal Model

Several studies of photodynamic technology (PDT) have shown stimulatoryeffects of PDT on cell types, especially the formation of collagenprecursor and enzymes associated with cellular development. The presentinvention offers means of skin rejuvenation which can be used to treatlarge surface areas with no significant adverse effects.

A study was performed in Wistar rats to evaluate the effects of thecomposition of the present invention in the presence of light in ananimal skin model, using a skin flap model. Using a template of 3 cm by9 cm (3×9) in plexiglas, a flap was traced on the dorsal skin with asurgical marker, taking as limits the inferior angles of the scapulaeand the superior bones of pelvis. A pure random pattern flap withcranial base was cut using sterile techniques and elevated through deepfascia, including superficial fascia, panniculus carnosus, thesubcutaneous tissue and skin. To minimize skin contraction andsimulating the human skin, a 0.5 cm subcutaneous layer of panniculuscarnosus from the wound edges was removed. During one hour, animpermeable barrier (e.g. a silicone sheeting) was placed between theflap and its donor site to eliminate the possibility of wound bedsupport. The sheet was then removed, the flap returned to its originalposition and the flap edges were surgically closed using 4/0 nylonsuture in an interrupted fashion. Immediately following flap closure,flap pedicle were coated with 13.5 g of gel formulation (0.5 g/cm²) andirradiated. The controls did not receive any treatment. Care was takento distribute ointment evenly along the entire flap. Gel formulation wasprepared the same day of the experiment. For Gel+Light group the animalswere treated with gel formulation, the flap was irradiated for 3 minuteswith a LED lamp.

Now referring to FIG. 3, the results show increased dermal thickness andnew collagen formation on skin treated with the composition of thepresent invention versus untreated skin. These result support the roleof the composition of the present invention in the stimulation of betteroutcomes in the skin rejuvenation process.

The embodiments and examples presented herein are illustrative of thegeneral nature of the subject matter claimed and are not limiting. Itwill be understood by those skilled in the art how these embodiments canbe readily modified and/or adapted for various applications and invarious ways without departing from the spirit and scope of the subjectmatter disclosed claimed. The claims hereof are to be understood toinclude without limitation all alternative embodiments and equivalentsof the subject matter hereof. Phrases, words and terms employed hereinare illustrative and are not limiting. Where permissible by law, allreferences cited herein are incorporated by reference in their entirety.It will be appreciated that any aspects of the different embodimentsdisclosed herein may be combined in a range of possible alternativeembodiments, and alternative combinations of features, all of whichvaried combinations of features are to be understood to form a part ofthe subject matter claimed.

1.-42. (canceled)
 43. A method for reversing or mitigating skin agingcomprising: a) topically applying on an area of a patient's skin to betreated a composition comprising at least one oxidant and at least onefluorescent dye capable of activating the oxidant; and b) illuminatingthe applied composition with actinic light for a period of about 60seconds to about 5 minutes per cm² of the area to be treated; therebyreversing or mitigating skin aging.
 44. The method according to claim43, wherein the actinic light has an emission wavelength of betweenabout 400 nm and about 600 nm.
 45. The method according to claim 43,wherein the actinic light has a power density of between about 900mW/cm² to about 2000 mW/cm².
 46. The method according to claim 43,wherein the actinic light is in continuous motion over an area to betreated.
 47. The method according to claim 43, wherein the at least oneoxidant is selected from at least one of hydrogen peroxide, carbamideperoxide and benzoyl peroxide.
 48. The method according to claim 43,wherein the composition further comprises at least one hydrophilicgelling agent.
 49. The method according to claim 48, wherein the atleast one hydrophilic gelling agent is selected from at least one ofglucose, modified starch, methyl cellulose, carboxymethyl cellulose,propyl cellulose, hydroxypropyl cellulose, a carbomer polymer, alginicacid, sodium alginate, potassium alginate, ammonium alginate, calciumalginate, agar, carrageenan, locust bean gum, pectin and gelatin. 50.The method according to claim 43, wherein the composition furthercomprises at least one chelating agent selected fromethylenediaminetetraacetic acid (EDTA) an ethylene glycol tetraaceticacid (EGTA).
 51. The method according to claim 43, wherein thecomposition further comprises at least one of rhodamine B, rhodamine WT,rhodamine G, phloxine B, rose bengal, eosin B, fluorescein. ErythrosineB, saffranin O, basic fuchsine, acid fuchsine, 3,3′-dihexylocarbocyanineiodide, carminic acid, indocyanine green, crocetin, alpha-crocin(8,8-diapo-8,8-carotenoic acid), zeaxanthine, lycopene, alpha-carotene,beta-carotene, merbromine, bixin, fucoxanthine, methyl violet, neutralred, para red, amaranth, carmoisine, allura red AC, tartrazine, orangeG, ponceau 4R, methyl red, murexide-ammonium purpurate, pyronine Y andpyronine B.
 52. The method according to claim 43, wherein thecomposition further comprises at least one lipolysis stimulating factorselected from caffeine and paraxanthine.
 53. The method according toclaim 43, wherein the fluorescent dye is eosin Y.
 54. The methodaccording to claim 43, wherein the fluorescent dye is selected from axanthene derivative dye, an azo dye, a biological stain and acarotenoid.
 55. The method according to claim 43, wherein thecomposition further comprises at least one healing factor selected fromhyaluronic acid, glucosamine and allantoin.